The long term goal of this research is to understand a signal transduction pathway that regulates programs of gene expression by controlling the subcellular localization of regulatory proteins. In the fruit fly Drosophila, intracellular transduction of a signal received at the surface of the embryo promotes nuclear translocation of the transcription factor Dorsal and thereby establishes the dorsoventral axis. In flies, plants, and mammals, homologous pathways direct nuclear import of related transcription factors as a critical step in immune or defensive responses. In addition to Dorsal, the Drosophila dorsoventral signaling pathway requires the transmembrane receptor Toll, the scaffolding protein Tube, the protein kinase Pelle, and the inhibitor Cactus. The fact that these five pathway components have been characterized at both the genetic and molecular level and can be assayed by embryo microinjection makes this system particularly amenable to experimental investigation. It is now possible, therefore, to address fundamental questions about the mechanism for signal transduction. The first two specific aims of this proposal will focus on the initiation of intracellular signaling. The potential function of the actin-binding protein Filamin as a link between Toll and Tube will be investigated in coimmunoprecipitation and genetic interaction studies. Additional experiments will address the mechanism and significance of Tube relocalization in response to Toll activation. The third and fourth aims will address how the signal is propagated. Autophosphorylation will be explored as a potential mechanism for Pelle regulation in vivo. Molecular and biochemical studies will also be carried out to determine which pathway proteins undergo Pelle-mediated phosphorylation in embryos. The fifth and final specific aim will focus on the spatial regulation of signaling within the syncytial environment of the blastoderm embryo. Given the conserved nature of the signaling pathway, the results of the proposed research should be of substantial interest with regard not only to pattern formation in the insect embryo, but also to signal transduction and immunity in a broad range of plants and animals.